ZONE PAPER ELECTROPHORESIS STUDIES ON RADIOIODINATED HUMAN SERUM ALBUMIN 1
By ELEMER R. GABRIELI, DICRAN GOULIAN, JR., THORN KLNERSLY,2 AND
RAYMOND COLLET 3
(Fromn the Sectioni of Medical Physics, Departmiienits of Physiology anid Initcrilal Medicile,
Yale University School of Medicinie, Newc, Haveni, Conn.)
(Submitted for publication April 22, 1953; accepted October 7, 1953)
INTRODUCTION
lodination of human serum albumin has been
investigated considerably. A careful report and
review of the literature on physico-chemical aspects was given recently by Hughes and Straessle
(1). These authors state: "The experiments reported indicate that considerable amounts of iodine, varying from very small amounts, to amounts
greater than the equivalent of the tyrosol residues, may readily be introduced into human serum albumin without detectable changes...
A further discussion of the properties of these
iodoproteins is given. Under similar conditions
of preparation, radioactive ( 131 ) iodinated human
serum albumin (IHSA) could be expected to display properties consonant wvith the above investi-
gation.
A commercial IHSA preparation
4
has become
available recently and was used during investigations in this laboratory concerning vascular damage following small doses of ionizing radiation
(2). The IHSA disappearance rate from the
circulation was established in control rats and
compared with that of irradiated animals; the
effects of the ionizing radiation were indicated by
the altered IHSA disappearance rate from the
vascular bed. In these experiments, it was noted
that the IHSA disappearance rate in control animals showed irregularities. In the first hour after
injection, the disappearance rate was faster than
expected. This observation suggested that an in1Aided in part by grants from the Atomic Energy
Commission, Contract No. AT (30-1)1068 and Eli Lilly
and Company.
2James Purcell Memorial Research Fellow, Department of Surgery.
3 Luria Foundation Fellow.
4 Obtained from Abbott Laboratories, North Chicago,
Illinois (Lot. Nos. A-254-24, B-21-27, B-210-41, and
B-233-1).
vestigation should be made on the physico-chemical properties of the IHSA preparation.
METHODS
Zone paper electrophoresis was utilized with the apparatus of Koiw, Wallenius, and Gronwall (3-5).
Runs were made using veronal buffer pH 8.6, ionic
strength of 0.05, 240 volts, Munktell No. 20 and Whatman No. 1 filter paper. After a varying migration time,
wet paper strips were removed, placed in a 70° C. oven
to dry for an hour and subsequently stained with
bromphenol blue (3).
-Radioautography was accomplished by placing the
filter paper strip against Kodak Medical X-ray film
(tinted safety base, duplitized) and arranging the two
strips between pieces of plate glass which were compressed by weights during the period of exposure.
Identification marks were pricked through both strips
to permit reconstruction of their relative positions after
completion of radioautography. The film was developed
in Kodak D-19 developer for four minutes, fixed in
Kodak Acid Fixer twice the clearing time, and washed
in water for one hour.
Measurements of radioactivity, were carried out by
two different methods. In some experiments the filter
paper was cut across into 5 mm. strips which were then
cut into three pieces and placed, all with the same side
up, in a planchette under a thin mica end-window type
Geiger-Mueller tube (TGC-1). All radioactivity was
measured with a scaler (Tracerlab Autoscaler) with a
counting accuracy of better than ± 2 per cent.
For measuring the radioactivity in other experiments,
a special device designed in this laboratory 5 was employed (Figure 1). A filter paper strip was fixed at
both ends to a flat, thin brass plate which was attached to
a threaded screw. Rotating a connected circular handle
advanced the brass plate, and the paper strip, 0.05 inch
per turn. This brass plate and turning screw assembly
was bolted to a lead shield (Tracerlab). A separate
part, containing an adjustable slit and made of 7 mm.
thick iron, was centered with respect to the GeigerMueller tube and placed at 1 mm. distance above the
brass plate. Width of the slit was 2 mm. and its length
equalled the width of the brass plate. This part was also
bolted to the lead shield.
Built by Van Collie Studios, Wilton, Connecticut.
136
w_
PAPER ELECTROPHORESIS OF RADIO-IODINATED SERUM ALBUMIN
137
K
J
H
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F
E
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FIG. 1. CONSTRUCTION OF SCANNING DEVICE FOR MEASURING
RADIOACTIVITY ON A PAPER STRIP
A-lock handle; B-rotating disk; C-lock notch; D-stable
disk; E-frame; F-moving block; G-brass plate; H- stabilizing rods; I-screw; J-stable block; K-adjustable slit.
Protein-bound dye determinations were made, after
the radioactivity measurements, by cutting the filter paper strips into 5 mm. sections, placing these strips in
test tubes and eluting the dye in 5 ml. of 5 per cent
sodium carbonate in 50 per cent methanol. After an
hour, the color intensities of the eluates were determined
in a Beckman spectrophotometer at 595 miu.
EXPERIMENTAL
Further experiments were performed to investigate this large amount of radioactivity which
continually appeared in the area usually occupied
by the globulins. Human blood serum and IHSA
were mixed as above, but the drop was pipetted
onto the middle of a filter paper strip. During
electrophoresis of these samples, the cathode and
anode were switched after three hours and the
Samples for zone paper electrophoresis were
prepared by mixing 1.0 ml. IHSA with 1.0 ml.
normal human blood serum and pipetting 0.04 ml.
RADIGCTIVITY
of this mixture onto Munktell No. 20 or Whatman No. 1 filter paper. Figure 2 indicates the results of this experiment. Radioactivity appears
increasingly detectable from the "starting point,"
i.e., the place where the drop was applied, to the
albumin fraction. The greatest amount of radio-PROTEIN
activity does not coincide with the localization of
the normal human serum albumin. In all of the
37 electrophoretic runs using four different
batches of IHSA, the peak of radioactivity appeared distinctly ahead of the normal human serum albumin.
In rats, 0.5 ml. IHSA was injected intravenously into two normal animals. Two hours
later, blood samples were taken, allowed to clot,
15
1
20 CM
5
IO
and centrifuged to obtain the serum for electroFIG. 2-a. ELECTROPHORETIC PATTERN ON MUNKTELL
phoresis. Figure 3 shows one of the results. The
picture appears identical to the IHSA-human No. 20 FILTER PAPER OF NORMAL HUMAN SERUM MIXED
WITH IHSA
serum experiment in that the IHSA is slightly
The arrows represent starting point and direction of
separated from the rat albumin, and an increasing migration.
Curve labeled "protein" shows concentraamount of radioactivity can be noted from the tion of bromphenol blue stain eluted from 0.5 cm. strips.
starting point to the albumin peak.
Other curve is radioactivity measured from same strips-
138
E. R. GABRIELI, D. GOULIAN, JR., T. KINERSLY, AND R. COLLET
I
-
*
I'
I
FIG. 2-b. RADIOAUTOGRAPH (UPPER) AND PICTURE (LOWER) OF A
SIMILAR EXPERINIENT
Arrows represent starting points. Vertical lines show limits of
bromphenol blue staining. Note increased migration and trailing of
IHSA on radioautograph.
run was continued for another nine hours. Figure 4 shows the results of this experiment.
Whereas the bromphenol blue staining appears
as usual, the radioactivity curve is quite different.
In this three hour-reverse current-nine hour electrophoresis, there is a peak of radioactivity at each
end of the pattern and radioactivity between the
two peaks seems greatly diminished.
Hence, it was apparent that IHSA might contain at least two distinct fractions. This sup-
R.ADIMTWY
RADIOACTI\ATY
wv~~0
-PROTEIN
PROTEIN
~~ ?HOUR
IJ
5
10
FIG. 3. ELECTROPHORETIC PATTERN ON MUNKTELL No.
20 FILTER PAPER OF RAT BLOOD SERUM
IHSA was inj ected intravenously two hours before
blood sample was taken. Arrows show starting point
and direction of migration. For curves labeled "protein"
and "radioactivity" see Figure 2-a.
3HOURSI
CM
FIG. 4-a. ELECTROPHORETIC PATTERN ON MUNKTELL
No. 20 FILTER PAPER OF NORMAL HUMAN SERUM
MIXED WITH IHSA
Arrows indicate the starting point, the initial three
hour migration and then nine hour migration in the opposite direction. Curve labeled "protein" shows concentration of bromphenol blue stain eluted from 0.5 cm.
strips. Other curve is radioactivity measured from same
strips.
139
PAPER ELECTROPHORESIS OF RADIO-IODINATED SERUM ALBUMIN
I.S
FIG. 4-b. RADIOAUTOGRAPH (UPPER) AND PICTURE (LOWER)
OF A SIMILAR EXPERIMENT
Arrows represent starting point. Vertical lines show limits of
bromphenol blue staining. Note considerable exposed area of
radioautograph to right of bromphenol blue pattern.
position was tested by experimental attempts to
separate the two fractions.
To normal human serum, a very small amount
of bromphenol blue was added. In minute concentrations, only the albumin absorbs the dye, permitting one to visualize the locus of the albumin
during electrophoresis. A large drop of this dyecontaining sample was pipetted onto a Munktell
No. 20 filter paper strip, and the IHSA-human
serum mixture was placed on a similar paper
strip. These two different samples were arranged
alongside each other between the same pair of
buffer-containing cells, and the starting points
were aligned so as to be equi-distant from the
cells. After six hours, the filter paper strips were
removed, the starting points realigned, and, using
the albumin-bromphenol blue migration as a
guide, the albumin section of the IHSA strip was
~
I4
RAD~OACTMTY
fi
4X
:9
t.
ta
.
0
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0
0
I
I
2
3
9
4
NM
ftm.
S 5
FIG. 6. SECOND ELECTROPHORESIS ON MUNKTELL No.
20 FILTER PAPER OF FLUID OBTAINED FROM ALBUMIN
SECTION OF A FIRST RUN
FIG. 5. DEVICE FOR REMOVING FLUID FROM
PAPER STRIP (SEE TEXT)
A FILTER
Arrows represent starting point and direction of migration. Curve is radioactivity as measured with scanning device (Figure 1).
140
E. R. GABRIELI, D. GOULIAN, JR., T. KINERSLY, AND R. COLLET
2
3
9
HOURS '3HOURS
8
INES
FIG. 7. ELECTROPHORETIC PATTERN ON WHATMAN
No. 1 FILTER PAPER OF NORMAL HUMAN SERUM MIXED
WITH IHSA
Compare radioactivity with Figure 4-a.
Immediately the section was placed in
centrifuge tube containing a mushroom-shaped
piece of plastic with six holes through the top
(Figure 5). Centrifuging enabled one to separate about 0.5 ml. of fluid from the filter paper.
A second electrophoretic run was made on this
fluid and the strip was measured for radioactivity
using the scanning device (Figure 1). The results of this experiment are indicated in Figure 6.
In this attempt to separate the albumin from the
"trailing" fraction the albumin was retained, but
the trailing fraction was almost eliminated.
An experiment was made to determine whether
the degree of trailing was consistent with or dependent on the type of filter paper used. When
Whatman No. 1 filter paper was used in three
hour-reverse current-nine hour electrophoresis,
no peak could be noted in the three hour-direction
and the trailing in the nine hour direction was almost absent (Figure 7).
cut out.
a
DISCUSSION
The increased mobility of IHSA is perhaps due
some minute change in the charge or shape of
the albumin molecule. The trailing, however, reveals another interesting difference between the
IHSA and the human blood serum. In the exto
periment in which the direction of migration was
changed after three hours and the IHSA-normal
serum mixture was allowed to move in the opposite direction for another nine hours (Figure
4), no evidence of normal human serum proteins,
when measured by the amount of bromphenol
blue staining, was demonstrable in the area of
the first three hour migration. On the other hand,
the IHSA trailing, when measured by its radioactivity, displayed a much different picture. Here
a definite peak of radioactivity was noticeable in
the three hour direction, and the trailing in the
nine hour direction was greatly diminished.
This experiment ruled out the possibility that
trailing represents contamination by some radioactive globulins which may have been present in
IHSA from the beginning or which may have occurred during the mixing with normal human serum. If some alpha or beta globulins had contained any radioactivity, it should be measurable
on corresponding spots of the globulins, but the
two peaks of radioactivity were not in globulin
areas.
Additional evidence that IHSA contains at
least two fractions was obtained in the experiment
in which an attempt was made to separate the
albumin fraction of the IHSA from the trailing
fraction (Figure 6). The albumin peak was still
present, but the trailing fraction almost disappeared.
Another implication of this study concerns the
interpretation of trailing, viz., a) trailing is an
undesirable artifact, b) trailing represents an additional separation. In our experiments it was
shown that IHSA trailing on Munktell No. 20
filter paper appears to be a distinct and separable
fraction from the albumin. That this trailing
was barely evident on Whatman No. 1 filter paper
does not necessarily mean that this paper is less
effective in separation.
An analogous situation might be found in the
work of Kunkel and Slater (6, 7) who reported
trailing of beta lipoproteins along the path of migration on Whatman 3 MM filter paper, but they
found no trailing to occur when the filter paper
was replaced by a starch medium. During some
experiments on lipoproteins in our laboratory, it
was noticed that Munktell No. 20 filter paper.
stained with Sudan Black, also showed trailing.
PAPER ELECTROPHORESIS OF RADIO-IODINATED SERUM ALBUMIN
But it is felt that this represents a subdivision of
the beta lipoproteins into two parts, one being
the trailing, and the other being, a fraction which
migrates in the same manner as beta proteins.
It may now begin to appear that the choice of
an electrophoretic medium depends on that which
one wants to demonstrate. Use of a starch medium would seem advantageous in many physicochemical studies. But for the detection of a slight
inhomogeneity of a colloidal material, paper electrophoiesis may provide much useful information.
The biological meaning of changes in the albumin molecule due to the iodination process may be
difficult to evaluate from these electrophoretic
studies. Whether or not these results are significant enough to limit the use of the IHSA studied
in this paper cannot be definitely determined at
this time. However, when it is considered that
experiments in this laboratory indicated a disappearance of IHSA in the blood stream of rats
which did not seem to follow the expected rate,
it may be implied that the IHSA fractions have
different metabolic rates.
SUMMARY
Commercial iodinated (I131) human serum albumin was studied by means of zone paper electrophoresis. In four different samples, at least
two fractions were noticed. One showed a
slightly higher mobility than noniodinated serum
albumin; the other fraction appeared in the form
of trailing. The two fractions could be separated,
and the trailing consequently eliminated. Hence,
141
the trailing appears not to be an artifact. The
usefulness of paper electrophoresis in the detection of slight inhomogeneities of colloidal materials and the biological significance of the two
fractions were discussed.
ACKNOWLEDGM ENT
The authors wish to acknowledge a discussion of this
paper with Dr. Kenneth Sterling and a critical reading
of the manuscript by Dr. John P. Peters.
REFERENCES
1. Hughes, W. L., Jr., and Straessle, R., Preparation
and properties of serum and plasma proteins.
XXIV. Iodination of human serum albumin. J.
Am. Chem. Soc., 1950, 72, 452.
2. Gabrieli, E. R., Kinersly, T., and Goulian, D., Effect
of ionizing radiation on the capillary wall. To be
published.
3. Koiw, E., Wallenius, G., and Gronwall, A., L'utilisation clinique de l'electrophorese sur papier filtre,
comparaison avec l'electrophorese a tube en u selon
la methode de Tiselius. Bull. Soc. chim. biol.,
1951, 33, 1940.
4. Koiw, E., Wallenius, G., and Gronwall, A., Paper
electrophoresis in clinical chemistry. A comparison with Tiselius' original method. Scandinav. J.
Clin. & Lab. Invest., 1952, 4, 47.
5. Gronwall, A., On paper electrophoresis in the clinical
laboratory. Scandinav. J. Clin. & Lab. Invest.,
1952, 4, 270.
6. Kunkel, H. G., and Slater, R. J., Lipoprotein patterns
of serum obtained by zone electrophoresis. J.
Clin. Invest, 1952, 31, 677.
7. Kunkel, H. G., and Slater, R. J., Zone electrophoresis
in a starch supporting medium. Proc. Soc. Exper.
Biol. & Med., 1952, 80, 42.